Journal: The Journal of general virology

Article Title: Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

doi: 10.1099/jgv.0.001899

Figure Lengend Snippet: Fig. 4. Binding of the SARS-CoV-2 S1 product to the human ACE2 receptor. (a) Whole cell lysates (WCL) were prepared at 9 h p.i. from MA104 cells infected with the indicated rSA11 viruses. The lysates were incubated with hACE2-Fc, a recombinant protein consisting of the extracellular domain of human ACE2 fused to an IgG1 Fc tag. Protein-A spin columns were used to recover hACE2-Fc, and associated proteins, from the lysates. The recovered samples were examined by immunoblot assay using antibodies specific for S1 products (FLAG/His antibody, SARS CoV-2 S1 antibody (ABclonal A20136). The red arrow head identifies S1 protein recovered by the pulldown assay. MWM, molecular weight markers. (b) The residual S1 proteins in the flow through fractions of the protein-A spin columns was determined by immunoblot assay using FLAG/His antibody. The same blot was re-probed with antibodies specific for SARS CoV-2 S1 (αS1/ABclonal), rotavirus VP6, and cellular β-actin.

Article Snippet: Membranes were blocked with phosphate- buffered saline containing 5 % non- fat dry milk and probed with rabbit polyclonal SARS- CoV- 2 S1 antibody (ABclonal A20136, 1 : 1000 dilution), mouse monoclonal (IgG1) SARS- CoV- 2 S1 antibody (R and D Systems MAB105403, 1 : 250), guinea pig polyclonal NSP3 (NIH Lot 55068, 1 : 2000 dilution) or VP6 (NIH Lot 53963, 1 : 2000) antisera, mouse monoclonal FLAG M2 (F1804, Sigma- Aldrich, 1 : 2000) or His- tag antibody (MCA1396, Bio- Rad, 1 : 1000), or rabbit monoclonal β-actin antibody (D6A8, Cell Signalling Technology, 1 : 1000).

Techniques: Binding Assay, Infection, Incubation, Recombinant, Western Blot, Molecular Weight

Journal: The Journal of general virology

Article Title: Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

doi: 10.1099/jgv.0.001899

Figure Lengend Snippet: Fig. 5. Localization of SARS CoV-2 S1 protein in rSA11/S1f-infected cells. MA104 cells were mock infected or infected with rSA11/S1f and, at 9 h p.i., fixed with ice cold methanol. Afterwards, the cells were incubated with rabbit S1/ABclonal antibody, mouse NSP2 or NSP4 antibody, followed by Alexa 488 anti-rabbit IgG (green) and Alexa 594 anti-mouse IgG (red) to determine the locations of SARS-CoV-2 S1 and rotavirus NSP2 and NSP4 proteins. Nuclei were detected by staining with DAPI. Cells were analysed with a Nikon Eclipse NiE microscope (100× oil immersion objective) and images were captured with a Hamamatsu Orca-Flash 2.8 sCMOS high resolution camera. Images include 20 µm size bars.

Article Snippet: Membranes were blocked with phosphate- buffered saline containing 5 % non- fat dry milk and probed with rabbit polyclonal SARS- CoV- 2 S1 antibody (ABclonal A20136, 1 : 1000 dilution), mouse monoclonal (IgG1) SARS- CoV- 2 S1 antibody (R and D Systems MAB105403, 1 : 250), guinea pig polyclonal NSP3 (NIH Lot 55068, 1 : 2000 dilution) or VP6 (NIH Lot 53963, 1 : 2000) antisera, mouse monoclonal FLAG M2 (F1804, Sigma- Aldrich, 1 : 2000) or His- tag antibody (MCA1396, Bio- Rad, 1 : 1000), or rabbit monoclonal β-actin antibody (D6A8, Cell Signalling Technology, 1 : 1000).

Techniques: Infection, Incubation, Staining, Microscopy

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: Viral findings in mild versus severe SARS-CoV2 infection. The control lungs showed no viral protein or RNA (panel A) whereas the lung tissue from someone who died of COVID-19 showed high viral RNA (panel B) and the spike protein subunit 2 (panel C) in the same alveolar wall distribution. Similarly, note the absence of the viral spike protein in the uninfected nasopharynx swab (panel D) and the strong signal for spike subunit 1 (panel E) and spike subunit 2 proteins (panel F) in glandular cells from a person with mild disease. NP – nasopharyngeal, NL – normal control, and the signal is brown due to DAB with hematoxylin counterstain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection, Control

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: Compilation of the immune response to mild infection in the nasopharyngeal swabs. a

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: Compilation of the immune response to fatal infection in the COVID-lungs. a

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection, Virus

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: Immune response in mild versus fatal SARS-CoV2 infection. The figure shows a graphic representation of the fold changes from baseline in the nasopharyngeal swabs positive for SARS-CoV2 (blue columns) as well as the lung tissues from fatal COVID-19 that either had very high copy SARS-CoV2 (orange columns) or were not associated with SARS-CoV2 infection (gray columns). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: In situ demonstration of the immune response in mild versus fatal SARS-CoV2 infection. Note the absence of a CD3 response in the uninfected nasopharynx swab (panel A) and the strong infiltration by these cells in an infected swab from a person with mild disease (panel B). Similarly, the control nasopharynx sample shows no PDL1 expression (panel C) whereas the infected nasopharynx shows a strong PDL1 response (panel D). Note that the lung from a fatal COVID-19 case not associated with viral infection did show a strong CD8 response (panel E) whereas this lung tissue from someone who died of COVID-19 that was strongly positive for SARS-CoV2 did not show a CD8 response (panel F). The control lungs showed a very weak PDL1 signal (panel G) whereas the COVID-19 lungs with high viral copy number did show strong PDL1 expression (panel H). NP – nasopharyngeal, NL – normal control, and the signal is brown due to DAB with hematoxylin counterstain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: In Situ, Infection, Control, Expressing

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: In situ demonstration of the clotting and complement activation in the fatal COVID-19 lungs. Note the absence of complement component 6 in the normal lung (panel A) as well as the lung from a fatal COVID-19 case that did not contain detectable SARS-CoV2 RNA (panel B). Panel C shows that the signal for complement component 6 in the COVID-19 lung tissue associated with high SARS-CoV2 copy number localizes only to the alveolar septa (arrows) which also shows a strong signal for fibrinogen (panel D); no signal is evident in the small vessels (oval). Panel E shows the lack of signal for CD41 in the normal lung controls. In comparison, note the CD41+ platelet aggregates in the small vessels in a lung from a person who died of COVID-19 associated with high viral copy number (panel F) as well as from the same lung that was not associated with SARS-CoV2 infection (panel G). However, note that the CD41 signal localizes only to the alveolar septa in the lung with high copy SARS-CoV2 (arrow, panel F). Co-localization of CD41 (fluorescent red) with SARS-CoV2 RNA (fluorescent green) documents that CD41 strongly co-expressed with the viral RNA in the lung samples with high copy viral RNA (seen as fluorescent yellow, panel H). NL – normal control, and the signal in panels A–D is brown due to DAB and fast red in panels E–G with hematoxylin counterstain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: In Situ, Coagulation, Activation Assay, Comparison, Infection, Control

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: Correlation of the H&E findings with the viral load in fatal COVID-19. The figure shows a graphic representation of the histologic findings in the lung tissues from people who died of COVID-19 where there was high viral copy number (orange columns) and those not associated with direct SARS-CoV2 infection (blue columns). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection

Journal: Annals of Diagnostic Pathology

Article Title: The differential immune response in mild versus fatal SARS-CoV2 infection ☆

doi: 10.1016/j.anndiagpath.2022.152032

Figure Lengend Snippet: H&E findings of mild versus fatal SARS-CoV2 infection. Panels A–C compare the cytologic findings in the normal nasopharynx (panel A) and mild infection (panel B); note that the glandular cells show degenerative changes that is highlighted with the EMA immunohistochemistry test (panel C). Panels D–F show successively high magnifications in a lung from a person who died of COVID-19 in which viral RNA was not evident; note the presence of extensive organizing pneumonia with scattered chronic inflammatory infiltrates. Panels G-I show successively high magnifications in a lung from a person who died of COVID-19 with high copy viral RNA; note the diffuse alveolar damage characterized by hyaline membrane formation, hemorrhage, and destruction of lining pneumocytes.

Article Snippet: SARS-CoV2 specific antibodies against nucleocapsid (catalogue #9099), spike subunit 1 (#9083), and spike subunit 2 (#9123) were from ProSci (Poway, CA).

Techniques: Infection, Immunohistochemistry, Membrane

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: HSF1A leads to a reduction in the levels of ADP-ribosylated Gαi within cells, while it has no impact on the enzymatic activity of PTS1. ( a ) CHO cells were subjected to pre-incubation with either HSF1A or VER for 1 h at 37 °C. In comparison, untreated cells (con) serve as the control. For further control, cells were treated with the solvent of both inhibitors, DMSO, at concentrations corresponding to the 200 µM HSF1A. Following this, the cells were exposed to 10 ng/mL PT for 4 h, both with and without the respective inhibitors. The cellular lysates were then examined to determine the ADP-ribosylation status of Gαi through incubation with 100 nM PTS1 in the presence of biotin-NAD + . The presence of biotin-labeled (ADP-ribosylated) Gαi was detected, and the results were normalized to the loading control, confirmed by Hsp90 detection. The values displayed indicate the signal percentage relative to untreated cells, and are normalized to the respective loading control, with mean ± SEM values obtained from at least five values across five independent experiments. Significance is evaluated using mixed-effects analysis and Dunnett’s multiple comparisons test, with values compared to samples treated solely with PT (white bar). ( b ) A549 cells were pre-incubated with HSF1A, or DMSO (corresponding to 200 µM HSF1A) as the control, for 30 min at 37 °C. Similar to panel ( a ), 10 ng/mL PT was introduced to the cells for a 4 h period in the presence, or absence, of the inhibitors. The lysates were then analyzed to assess the ADP-ribosylation status of Gαi. Comparable protein loading was again confirmed by Hsp90 detection, and the results were expressed as the signal percentage relative to untreated cells, normalized to the Hsp90-based loading control. Mean ± SEM values from no less than five values across five independent experiments are provided, with significance determined through mixed-effects analysis and Dunnett’s multiple comparisons test, referring to samples treated only with PT (white bar). ( c ) Recombinant Gαi was pre-incubated with either HSF1A, or DMSO as the control, for 30 min at room temperature. For further control, recombinant Gαi was also incubated with buffer exclusively. Subsequently, 100 nM PTS1 and 10 μM biotin-labeled NAD + were added, and incubation was carried out for 30 min at room temperature. The presence of biotin-labeled (ADP-ribosylated) Gαi was identified using streptavidin–peroxidase, and signal intensities were quantified via densitometry. The values are presented as a percentage relative to samples treated solely with PTS1, with mean ± SEM values from no less than four values across four independent experiments. Significance is determined through mixed-effects analysis and Dunnett’s multiple comparisons test, referring to samples treated only with PTS1 (white bar). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Activity Assay, Incubation, Comparison, Control, Solvent, TNKS1 Histone Ribosylation Assay, Labeling, Recombinant

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: HSF1A does not inhibit the binding of PT to cells. ( a ) CHO cells were subjected to a 30 min pre-incubation with either HSF1A or VER, while DMSO was employed as a control, all carried out at 37 °C. Subsequently, cells were exposed to a total of 500 ng/mL PT at a temperature of 4 °C for a duration of 40 min. After thorough washing, the presence of bound PT was detected using Western blotting and a specific PTS1 antibody. Assurance of equivalent loading was validated through Hsp90 detection. The Western blot signals were quantified and then normalized both to the Hsp90 signals and to the samples treated solely with PT (mean ± SEM values), derived from a minimum of four values across four independent experiments. Statistical significance was assessed using mixed-effects analysis and Dunnett’s multiple comparisons test, with the values referring to samples treated exclusively with PT being represented by a white bar. ( b ) A suspension of CHO cells underwent a 30 min pre-incubation with HSF1A or DMSO at 37 °C. Subsequently, the cells were cooled on ice and then exposed to 500 ng/mL PT labeled with 488-dye for a duration of 15 min under ice-cold conditions. After undergoing two wash cycles, the attached PT was identified using flow cytometry. The values presented are indicated as x-fold relative to the control. The dataset comprises 10 values from five separate experiments. Statistical analysis involved a one-way ANOVA test, with the results compared to samples treated exclusively with PT. **** p < 0.0001, ns = not significant.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Binding Assay, Incubation, Control, Western Blot, Derivative Assay, Suspension, Labeling, Flow Cytometry

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: When cells are treated with HSF1A, a reduced amount of PTS1 signal is observed within cells. CHO cells were subjected to a 30 min pre-incubation with HSF1A or VER at 37 °C. For control, cells were left untreated. Following this, PT at a concentration of 100 ng/mL was introduced for a 4 h period. After washing, the cells underwent fixation and permeabilization, followed by staining of PTS1 through incubation with a specific primary antibody, succeeded by a secondary fluorescence-labeled antibody. Nuclei were stained using Hoechst dye, and images were captured in a random fashion using a Keyence fluorescence microscope. The PTS1 signal was quantified from uncropped images (40× objective) and values are shown as percent of PT-only treated cells (mean ± SEM, n = 30 (30 images from three independent experiments). An average of 41.73 ± 1.019 (mean ± SEM) cells were captured per image. Statistical analysis involved a mixed-effects analysis and Dunnett’s multiple comparisons test, with the results compared to samples treated exclusively with PT. **** p < 0.0001. Green = PTS1, blue = nucleus. White squares indicate the magnified areas. Scale bar = 50 µm.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Incubation, Control, Concentration Assay, Staining, Fluorescence, Labeling, Microscopy

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: Interaction of TRiC/CCT subunit CCT5 with PTS1 in cells. A549 cells were pre-incubated with 200 µM HSF1A or the corresponding amount of its solvent, DMSO, for 30 min. Cells were challenged with 100 ng/mL PT for 4 h. Then, cells were carefully washed, fixed, and processed for a fluorescence-based proximity ligation assay (PLA) in accordance with the manufacturer’s guidelines. The cell nuclei were stained with Hoechst (blue). The resulting PLA signals (depicted in white) indicate instances of protein interaction between PTS1 and CCT5. White squares indicate the magnified areas below. The quantification of these signals per cell number is presented in the bar graph. The values are reported as mean ± SEM (n = 40 uncropped images captured in a random fashion using a Keyence fluorescence microscope 40× objective per condition from four independent experiments). An average of 37.09 ± 0.5213 (mean ± SEM) cells were captured per image. Significance levels tested using one-way ANOVA with Dunnett’s multiple comparisons test are indicated by asterisks (**** p ≤ 0.0001, ns denotes non-significant) and were tested against untreated control samples. Scale bar = 25 µm.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Incubation, Solvent, Fluorescence, Proximity Ligation Assay, Staining, Microscopy, Control

Journal: Toxins

Article Title: The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin

doi: 10.3390/toxins16010036

Figure Lengend Snippet: Schematics of the role of host cell chaperones in pertussis toxin activity. Pertussis toxin (PT) consists of the enzyme subunit PTS1 and the B-subunit pentamer, which facilitates cell binding. The cell binding is followed by endocytosis and retrograde transport of the toxin through the Golgi to the endoplasmic reticulum (ER). In the ER, PTS1 is released from the B-pentamer, unfolded, and transported to the cytosol by the ER-associated degradation pathway. This translocation and subsequent refolding of PTS1 is supported by several chaperones, Hsp90, Hsp70, cyclophilins (Cyps), and FK506 binding proteins (FKBPs), as well as the chaperonin TRiC/CCT complex.

Article Snippet: The samples were subjected to analysis using SDS–PAGE and Western blotting, with the specific antibody (Santa Cruz, #sc-57639 (63.1G9)) employed to detect PTS1.

Techniques: Activity Assay, Binding Assay, Translocation Assay